Midkine (hereinafter sometimes abbreviated as “MK” as required) is a growth/differentiation factor that was first discovered as a gene product expressed transiently in the process of differentiation induction of embryonic tumor cells (EC) with retinoic acid, being a polypeptide having a molecular weight of 13 kDa, rich in basic amino acids and cysteine.
The steric structure of MK has been determined by NMR and reported. When characterized structurally, MK is configured mainly with two domains. Specifically, MK consists of a fragment on the N-terminal side consisting of amino acid residues 1 to 52 (hereinafter referred to as “the N-fragment”), a fragment on the C-terminal side consisting of amino acid residues 62 to 121 (hereinafter referred to as “the C-fragment”) and a loop region that connects the fragments (amino acid residues 53 to 61). In the MK molecule, each of the N-fragment and the C-fragment has a steric structure consisting mainly of three reversed β sheet structures (hereinafter referred to as “domains”; a domain consisting of the amino acid residues 15 to 52 in the N-fragment referred to as “the N-domain”, a domain consisting of the amino acid residue 62 to 104 in the C-fragment referred to as “the C-domain”), and freely moving structures assuming no particular structure (hereinafter referred to as “tails”; a tail consisting of the amino acid residues 1 to 14 in the N-fragment referred to as “the N-tail”, and a tail consisting of the amino acid residues 105-121 in the C-fragment referred to as “the C-tail”). Bound to the outside of each domain is a tail that is rich in basic amino acids.
Known receptors of MK include receptor-type protein tyrosine phosphatase ζ (PTPζ), LRP (low density lipoprotein receptor-related protein), ALK (anaplastic leukemia kinase), integrin and syndecan and the like. MK is a highly positively charged protein containing large amounts of the basic amino acids lysine (K) and arginine (R). It has a heparin-binding site in the C-domain thereof, and is known to bind strongly to negatively charged molecules such as heparin and chondroitin sulfate E.
MK is a protein important in the developmental process, and is strongly expressed in midembryo. In contrast, expression in adults is limitative, and MK expression is found in vascular endothelium and particular mucosal epithelium. When a tissue is damaged, MK expression at the site increases, or newly induced. The produced MK promotes survival and movement of cells, and further exhibits various biological activities such as cell proliferation, altered morphology, chemokine expression and the like.
MK is also related to cancer, and MK expression is known to increase in many of human cancers. Such phenomenon has been observed in a wide variety of cancers, including esophageal cancer, thyroid cancer, urinary bladder cancer, colorectal cancer, gastric cancer, pancreatic cancer, thoracic cancer, liver cancer, lung cancer, breast cancer, neuroblastoma, glioblastoma, uterine cancer, ovarian cancer, prostate cancer and Wilms' tumor. By comparison of each case of various carcinomas, MK expression increases in about 80% of the cases. It has been reported an increase in the expression of MK was found in all cases of Wilms' tumor developed by the deletion of WT1 cancer suppressive gene and tumor in the nerve system caused by the deletion of NF-1 cancer suppressive gene.
MK with increased expression is also considered to promote the survival and movement of cancer cells and facilitate neovascularization to help the advancement of cancer. In neuroblastoma, urinary bladder cancer, glioblastoma and the like, it is known that prognosis is poorer in cancer patients with high MK expression than cancer patients with low MK expression. In a cell line derived from human gastric cancer, there is a strong correlation between resistance to anti-cancer agent and high expression of MK. In the cells derived from human liver cancer, MK is deeply involved in the cell proliferation thereof, and is also known to inhibit apoptosis of the cells.
From such relationship between MK and cancer, simultaneously with the utilization of MK to tumor marker, the development of a therapeutic drug for cancer targeting MK is attracting attention. As a therapeutic drug for cancer, one suppressing an increase in the expression of MK is designed, and antibody, siRNA, antisense oligoDNA and the like to MK have been studied (non-patent documents 1-3).
In recent years, applications of RNA aptamers to medicaments, diagnostic agents, and test drugs have been drawing attention; some RNA aptamers have already been in clinical study stage or in practical use. In December 2004, the world's first RNA aptamer drug, Macugen, was approved as a therapeutic drug for age-related macular degeneration in the US. An RNA aptamer refers to an RNA that binds specifically to a target substance such as a protein, and can be prepared using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. In the SELEX, an RNA that binds specifically to a target substance is selected from an RNA pool with about 1014 different nucleotide sequences. The RNA structure used has a random sequence of about 40 residues, which is flanked by primer sequences. This RNA pool is allowed to be assembled with a target substance, and only the RNA that has bound to the target substance is collected using a filter and the like. The RNA collected is amplified by RT-PCR, and this is used as a template for the next round. By repeating this operation about 10 times, an RNA aptamer that binds specifically to the target substance can be acquired. There are already some reports on the aptamer for MK (patent documents 1-3, non-patent document 4).
Aptamer drugs, like antibody drugs, can target extracellular factors. With reference to many scientific papers and other reference materials in the public domain, aptamer drugs are judged to potentially surpass antibody drugs in some aspects. For example, aptamers often show higher binding force and higher specificity than do antibodies. Aptamers are unlikely to undergo immune elimination, and adverse reactions characteristic of antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), do not occur with the use of aptamers. From the aspect of delivery, since aptamers are about 1/10 of antibody in size, delivery of a drug to the object site is easier. Since aptamers are produced by chemical synthesis, various modifications can be made easily, reduction of cost by large-scale production is possible. Meanwhile, the blood half-lives of aptamers are generally shorter than those of antibodies; however, this property is sometimes advantageous in view of toxicity. These facts lead to the conclusion that even when the same molecule is targeted, aptamer drugs potentially surpass antibody drugs.